Process for the preparation of optically active 7-substituted 3-(2-aminopropyl)indole derivatives and intermediates therefor

ABSTRACT

Disclosed are a process for preparation of an optically active 7-substituted 3-(2-aminopropyl)indole compound and an intermediate therefor. In the above preparation process, 7-substituted indole is reacted with L- or DL-serine in the presence of a tryptophan-synthesizing enzyme originating in microorganisms to form corresponding 7-substituted L-tryptophan, and it is subjected, if necessary, to reduction, protection, exchange and elimination.

TECHNICAL FIELD

[0001] The present invention relates to a process for preparation of anoptically active 7-substituted-3-(2-aminopropyl)indole compound and anintermediate therefor. The above indole compound can be obtained byusing as a starting material, 7-substituted-tryptophan prepared byreacting 7-substituted indole with L-serine in the presence of amicroorganism or a cell lyzate thereof.

BACKGROUND ART

[0002] Many of compounds out of indole derivatives are under developmentas medicines since they have a very interesting biological activity, orsome compounds have already been used as medicines.

[0003] Such derivatives include a drug represented by a formula:

[0004] (wherein A and B represent at least one substituent including ahydrogen atom) which acts on each subclass of β-adrenergic receptor(WO96/16938, J. Med. Chem., 25, p. 670 to 679 (1982), British Patent No.861,428 and the like). Carbon at a 2-position of a 2-aminopropane partbonded to a 3-position of an indole ring in these compounds is anasymmetric center, and plural stereoisomers originating therein can bepresent. However, specific isomers in these compounds are desirable interms of a biological activity in many cases as commonly observed inbiologically active substances. In actuality, a configuration at a2-position in the part described above in a compound having aβ₃-adrenergic receptor stimulation which is described in WO96/16938 ispreferably either R- or S-. Accordingly, if it is intended to providethe indole derivative described above having a configuration in whichthe 2-aminopropane part described above is 2(R) or 2(S), the finalcompound or the synthetic intermediate having the 2-aminopropane partdescribed above has to be subjected to optical resolution.

[0005] On the other hand, proposed in WO96/16938 is a method in whichformed from a corresponding amine compound is a part of:

[0006] and in which formed from a corresponding oxirane compound orcarboxylic acid compound is a part of:

[0007] When employing such method, a desired optical isomer shall beable to be obtained as the final compound if the 2R- or 2S-isomer isused as the amine compound described above according to the purposes.

DISCLOSURE OF THE INVENTION

[0008] An object of the present invention is to provide 7-substitutedindoles which are optically active amine compounds capable of being asynthetic intermediate of the indole derivative described in WO96/16938.

[0009] The present inventors have paid attentions as one means forachieving the object described above to applicability of L-tryptophan asa natural substance which has an indole skeleton and is provided with anoptical activity. There has been proposed a wide variety of productionprocesses for L-tryptophan such as a fermentation process using aproducing microorganism thereof (for example, Japanese PatentApplication Laid-Open No. 140891/1984) and a microbiological conversionprocess. In general, in the above conversion process, indole is reactedwith L-serine or pyrubic acid under the action of a tryptophan synthaseor a tryptophanase to produce L-tryptophan (for example, Japanese PatentApplication Laid-Open No. 55188/1989, Japanese Patent ApplicationLaid-Open No. 255082/1990, WO89/03428, Japanese Patent ApplicationLaid-Open No. 103283/1996 and Japanese Patent Application Laid-OpenNo.103284/1996). However, it is neither described nor indicated in theseproduction processes for L-tryptophan whether or not these processes canbe used for producing an L-tryptophan derivative having a substituent onan indole ring. On the other hand, known is a production process inwhich an indole derivative obtained by substituting with CH₃, OCH₃ or OHin any of a 2-, 4-, 5-, 6- and 7-positions of indole and DL-glycericacid or glycerol are added to a fermentation broth of a bacterial strainsuch as a Corynebacterium genus and an Escherichia genus and reacted toproduce an L-tryptophan derivative having a substituent corresponding tothe above indole derivative (Japanese Patent Application Laid-Open No.20392/1974).

[0010] If a carboxyl group of 7-substituted tryptophan out of thesederivatives can efficiently be converted to the other group, variousoptically active 3- and 7-substituted indoles shall be able to beprovided. When a carboxyl group is chemically converted to the othergroup, a stage for reducing the carboxyl group has to usually pass, andtherefore it shall be necessary for conversion of the carboxyl groupthat a hydroxyl group at a 7-position of indole does not an adverseeffect on such reduction reaction and that it is protected by a groupwhich can readily and selectively be eliminated after prescribedreaction. A typical example of such protective group for a hydroxylgroup includes a benzyl group.

[0011] The present inventors have found that 7-substituted indole havingbenzyloxy (PhCH₂O—) or diethylaminocarbonylmethoxy (Et₂NCOCH₂O—) at a7-position which is a far more bulky group than a group of the indolederivative described in Japanese Patent Application Laid-Open No.20392/1974, that is, CH₃, OCH₃ or OH can be converted to correspondingL-7-substituted tryptophan by using a tryptophan-synthesizing enzymeoriginating in microorganisms and that a carboxyl group of the abovetryptophan can chemically be converted to the other various substituentsat a good efficiency.

[0012] That is, the present invention is based on such knowledges.

[0013] Hence, according to the present invention, provided is a processfor preparation of an optically active indole derivative of the Formula(I):

[0014] [wherein R₁ is a hydrogen atom, a lower alkyl group or a groupselected from the following (a) and (b):

[0015] (a) a group of the formula —(CH₂)_(m)—CHR_(a)R_(aa) (whereinR_(a) is a hydrogen atom, a lower alkyl group, a lower alkoxycarbonylgroup, a carboxyl group or a phenyl group which may be substituted (asubstituent thereof is a lower alkyl group, a lower alkoxy group, ahydroxy group or a halogen atom); R_(aa) is a lower alkoxycarbonylgroup, a carboxyl group or a phenyl group which may be substituted (asubstituent thereof is a lower alkyl group, a lower alkoxy group, ahydroxy group or a halogen atom); and m is an integer of 0 to 3) and

[0016] (b) a group of the formula —(CH₂)_(p)—R_(b) (wherein R_(b) is alower alkanoyl group, a hydroxy group, a cyano group or a mono- ordi(lower alkyl)aminocarbonyl group, and p is an integer of 1 to 4); R₂and R₃ are each a hydrogen atom, or either of them is a hydrogen atomand the other represents a lower alkanoyl group, a lower alkoxycarbonylgroup or an aralkyloxycarbonyl group, or together form a phthaloylgroup; and R₄ is a hydrogen atom, a halogen atom, a hydroxy group, alower alkylsulfonyloxy group or a phenylsulfonyloxy group which may besubstituted (a substituent thereof is a lower alkyl group, a loweralkoxy group or a halogen atom)], comprising the steps of:

[0017] a) reacting L- or DL-serine with 7-substituted indole of theFormula (II):

[0018] [wherein R_(1a) is a group (c) or (d) shown below:

[0019] (c) a group of the formula —(CH₂)_(m)—CHR_(a)′R_(aa)′ (whereinR_(a)′ is a hydrogen atom, a lower alkyl group or a phenyl group whichmay be substituted (a substituent thereof is a lower alkyl group, alower alkoxy group, a hydroxy group or a halogen atom); R_(aa)′ is aphenyl group which may be substituted (a substituent thereof is a loweralkyl group, a lower alkoxy group, a hydroxy group or a halogen atom);and m is an integer of 0 to 3) and

[0020] (d) a group of the formula —(CH₂)_(p)—R_(b)′ (wherein R_(b)′ is amono- or di(lower alkyl)aminocarbonyl group, and p is an integer of 1 to4)] in the presence of a tryptophan-synthesizing enzyme originating inmicroorganisms to form 7-substituted-L-tryptophan of the Formula (III):

[0021]  (wherein R_(1a) is the same as described above),

[0022] b) chemically reducing a carboxyl group of 7-substitutedL-tryptophan of Formula (III) thus formed to a methylol group,

[0023] c) protecting and modifying, if necessary, an amino group,

[0024] d) turning, if necessary, a hydroxy group of methylol describedabove into sulfonyl to form a corresponding lower alkylsulfonyloxycompound or a phenylsulfonyloxy compound which may be substituted (asubstituent thereof is a lower alkyl group, a lower alkoxy group or ahalogen atom),

[0025] e) halogenating it, if necessary, with alkali halide orhalogenating a hydroxy group of methylol described above directly or inthe presence of triphenylphosphine to form a corresponding halide,

[0026] f) converting, if necessary, the sulfonyloxy compound or thehalide obtained in d) or e) to methyl by hydrogenating in the presenceof a palladium catalyst or using a metal hydride salt,

[0027] g) eliminating, if necessary, the protective group of the aminogroup, and

[0028] h) subjecting, if necessary, the substituent of R₁ to exchangereaction.

[0029] The compound of Formula (I) thus provided or a compoundoriginating in the above compound is useful as a precursor or anintermediate of a bioactive substance. For example, when R₂ and R₃ inFormula (I) are hydrogen atoms, the final intended compound having aβ₃-adrenergic receptor stimulation or a precursor thereof can beprepared by reaction with:

[0030] according to the process described in WO96/16938 described above.

[0031] Also, according to the present invention, provided is anoptically active indole derivative of the Formula (IV):

[0032] [wherein R₁ is a hydrogen atom, a lower alkyl group or a groupselected from the following (a) and (b):

[0033] (a) a group of the formula —(CH₂)_(m)—CHR_(a)R_(aa) (whereinR_(a) is a hydrogen atom, a lower alkyl group, a lower alkoxycarbonylgroup, a carboxyl group or a phenyl group which may be substituted (asubstituent thereof is a lower alkyl group, a lower alkoxy group, ahydroxy group or a halogen atom); R_(aa) is a lower alkoxycarbonylgroup, a carboxyl group or a phenyl group which may be substituted (asubstituent thereof is a lower alkyl group, a lower alkoxy group, ahydroxy group or a halogen atom); and m is an integer of 0 to 3) and

[0034] (b) a group of the formula —(CH₂)_(p)—R_(b) (wherein R_(b) is alower alkanoyl group, a hydroxy group, a cyano group or a mono- ordi(lower alkyl)aminocarbonyl group, and p is an integer of 1 to 4); R₂and R₃ are each a hydrogen atom, or either of them is a hydrogen atomand the other is a lower alkanoyl group, a lower alkoxycarbonyl group oran aralkyloxycarbonyl group, or together form a phthaloyl group; and R₄′is a halogen atom, a hydroxy group, a lower alkylsulfonyloxy group or aphenylsulfonyloxy group which may be substituted (a substituent thereofis a lower alkyl group, a lower alkoxy group or a halogen atom)]. Thecompound of the Formula (IV) is the compound of the Formula (I) in whichR₄ is a group other than a hydrogen atom.

[0035] Further, according to the present invention, provided is7-substituted-L-tryptophan of the Formula (III):

[0036] (wherein R_(1a) is the same as described above).

BEST MODE FOR CARRYIYING OUT THE INVENTION

[0037] The respective atoms or groups constituting the compoundsrepresented by (I), (II), (III) and (IV) according to the presentinvention commonly mean the following unless otherwise described.

[0038] The “lower alkyl group” means a linear or branched alkyl grouphaving 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms regardless ofwhether it is an independent group or a part of a particular group andincludes, for example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl and tert-butyl. The “lower alkoxy group” is a groupin which an alkyl part comprises the lower alkyl group described aboveand in which an oxygen atom is covalently bonded to the above alkyl, andspecific examples thereof include methoxy, ethoxy, propoxy andisopropoxy. The “lower alkoxycarbonyl group” is a group formed fromalkoxy having the lower alkyl group described above in an alkyl part andcarbonyl, and specific examples thereof include methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl and tert-butoxycarbonyl.The “lower alkanoyl group” is an acyl group formed from the lower alkylgroup described above as an alkyl part and carbonyl, and specificexamples thereof include acetyl and propionyl.

[0039] The “halogen” or “halogen atom” means usually fluorine, chlorine,bromine and iodine but in the definition of R₄ in Formula (I), it isparticularly preferably iodine, bromine and chlorine.

[0040] The “phenyl group which may be substituted with lower alkyl,lower alkoxy, hydroxy or halogen” means a phenyl group which may besubstituted with the same or different, up to four groups of the loweralkyl, lower alkoxy, halogen and hydroxy each described above, andcapable of being given as specific examples thereof are phenyl,4-methylphenyl, 4-methoxyphenyl, 2,6-dimethylphenyl,3,5-dihydroxyphenyl, 2,3-dichlorophenyl and 2,6-dichlorophenyl.Accordingly, capable of being given as specific examples of R₁ in thecase where R₁ is represented by the formula —(CH₂)_(p)—R_(b) in Formula(I) and R_(b) is the phenyl group described above are benzyl, phenethyl,3-phenylpropyl, 4-phenylbutyl, 4-methylbenzyl and 4-methoxybenzyl. WhenR₁ is one of these groups, the corresponding compound can be subjectedto biochemical conversion reaction, though described later in details,while holding such groups. Among these groups, particularly a benzylgroup can readily be eliminated by hydrogenolysis and therefore isfavorably converted to the other groups.

[0041] Specific examples of the “mono- or di-lower alkylaminocarbonylgroup” include methylaminocarbonyl, dimethylaminocarbonyl,ethylaminocarbonyl, methylethylaminocarbonyl and diethylaminocarbonyl.Representative examples of R₁ in the case where R₁ is represented by theformula —(CH₂)_(p)—R_(b) and R_(b) is the mono- or di-loweralkylaminocarbonyl group described above includediethylaminocarbonylmethyl and dimethylaminocarbonylmethyl. When a partoriginating in the compound of Formula (I) according to the presentinvention has these groups, the final compound shows a good bioactivityin many cases (refer to WO96/16938).

[0042] Either of R₂ and R₃ can be a hydrogen atom, and the other can bethe lower alkanoyl group, the lower alkoxycarbonyl group or thearalkyloxycarbonyl group (for example, benzyloxycarbonyl) each describedabove. In these cases, R₂, R₃ and a nitrogen atom to which they arebonded correspond to a so-called protected amino group in one group.Further, R₂ and R₃ can together form a phthaloyl group. In this case,R₂, R₃ and a nitrogen atom to which they are bonded correspond as wellto a protected amino group in one group. When the compound of Formula(I) holds such protected amino group, the compound of Formula (I) isuseful as a synthetic intermediate which can be derived into the othercompounds via R₂ and/or R₄. On the other hand, when both of R₂ and R₃are hydrogen atoms, the compound of Formula (I) is useful as a syntheticintermediate which can be derived into the other compounds via the freeamino group.

[0043] —CH₂—R₄ in Formula (I) shall not be restricted and can be a groupwhich is derived directly or indirectly from a carboxyl group.Accordingly, the group of R₄ can be hydroxy, lower alkylsulfonyloxy,lower alkyl, lower alkoxy or phenylsulfonyloxy which may be substitutedwith a halogen atom, a halogen atom and a hydrogen atom. The “loweralkyl” in these groups has the same meaning as described above.Accordingly, specific examples of alkylsulfonyloxy includemethanesulfonyloxy, and specific examples of phenylsulfonyloxy which maybe substituted with lower alkyl include p-toluenesulfonyloxy.

[0044] Compounds comprising combinations shown in the following Table 1can be given as the compounds of Formula (I) comprising the preferredcombinations of the respective groups explained above. TABLE 1

Compound No. R₁ —NR²R³ R₄ = OH 1 Bz- —NH₂ 2 Bz- —NPht 3 Bz- —NHCOO^(t)Bu4 Et₂NCOCH₂— —NH₂ 5 Et₂NCOCH₂— —NPht 6 Et₂NCOCH₂— —NHCOO^(t)Bu 7PhCH₂CH₂— —NH₂ 8 PhCH₂CH₂— —NPht 9 PhCH₂CH₂— —NHCOO^(t)Bu 10 4-MePhCH₂——NH₂ 11 4-MePhCH₂— —NPht 12 4-MePhCH₂— —NHCOO^(t)Bu 13 4-MeOPhCH₂— —NH₂14 4-MeOPhCH₂— —Pht 15 4-MeOPhCH₂— —NHCOO^(t)Bu R₄ = OTos 16 Bz- —NH₂ 17Bz- —NPht 18 Bz- —NHCOO^(t)Bu 19 Et₂NCOCH₂— —NH₂ 20 Et₂NCOCH₂— —NPht 21Et₂NCOCH₂— —NHCOO^(t)Bu 22 PhCH₂CH₂— —NH₂ 23 PhCH₂CH₂— —NPht 24PhCH₂CH₂— —NHCOO^(t)Bu 25 4-MePhCH₂— —NH₂ 26 4-MePhCH₂— —NPht 274-MePhCH₂— —NHCOO^(t)Bu 28 4-MeOPhCH₂— —NH₂ 29 4-MeOPhCH₂— —NPht 304-MeOPhCH₂— —NHCOO^(t)Bu R₄ = I 31 Bz- —NH₂ 32 Bz- —NPht 33 Bz-—NHCOO^(t)Bu 34 Et₂NCOCH₂— —NH₂ 35 Et₂NCOCH₂— —NPht 36 Et₂NCOCH₂——NHCOO^(t)Bu 37 PhCH₂CH₂— —NH₂ 38 PhCH₂CH₂— —NPht 39 PhCH₂CH₂——NHCOO^(t)Bu 40 4-MePhCH₂— —NH₂ 41 4-MePhCH₂— —NPht 42 4-MePhCH₂——NHCOO^(t)BU 43 4-MeOPhCH₂— —NH₂ 44 4-MeOPhCH₂— —NPht 45 4-MeOPhCH₂——NHCOO^(t)Bu R₄ = H 46 Bz- —NH₂ 47 Bz- —NPht 48 Bz- —NHCOO^(t)Bu 49Et₂NCOCH₂— —NH₂ 50 Et₂NCOCH₂— —NPht 51 Et₂NCOCH₂— —NHCOO^(t)Bu 52PhCH₂CH₂— —NH₂ 53 PhCH₂CH₂— —NPht 54 PhCH₂CH₂— —NHCOO^(t)Bu 554-MePhCH₂— —NH₂ 56 4-MePhCH₂— —NPht 57 4-MePhCH₂— —NHCOO^(t)Bu 584-MeOPhCH₂— —NH₂ 59 4-MeOPhCH₂— —NPht 60 4-MeOPhCH₂— —NHCOO^(t)Bu

[0045] The compounds described above are provided as compounds whichhave at least one asymmetric carbon in a molecule and which aresubstantially optically pure. The term “substantially optically pure”means that the compounds having a specific configuration in which acarbon atom combined with a NR₂R₃ group is an asymmetric center have apurity of 90% or more, preferably 95% or more.

[0046] The substantially optically pure compound of Formula (I)described above can efficiently be obtained by reacting 7-substitutedindole with serine in the presence of a tryptophan-synthesizing enzymeaccording to the present invention to produce correspondingL-7-substituted tryptophan (hereinafter referred to as a biochemicalconversion step) and subjecting L-7-substituted tryptophan thus obtainedwhich is used as a starting material to publicly known chemicalreaction.

[0047] According to the biochemical conversion step described above, theconversion reaction is carried out in the presence of atryptophan-synthesizing enzyme originating in microorganisms. The aboveterm “in the presence of a tryptophan-synthesizing enzyme” means asystem or an environment in which corresponding 7-substitutedL-tryptophan can be prepared from 7-substituted indole of Formula (II)and L-serine:

[0048] (wherein R_(1a) is the same as described above) by virtue of theaction of the tryptophan-synthesizing enzyme (or a tryptophan synthase:EC 4. 1. 99. 1.). Capable of being given as such system or environmentis a culture of a microorganism producing a tryptophan-synthesizingenzyme or a cell lyzate originating in the culture, which has anactivity for producing 7-benzyloxy-L-tryptophan from at least, forexample, 7-benzyloxyindole and L-serine. Such activity may allow theforming reaction described above to resultingly proceed regardless ofdepending on the action of any of complex (for example, α₂β₂, β₂ and thelike) of the respective subunits in the tryptophan-synthesizing enzyme(or a tryptophan synthase: EC 4. 1. 99. 1.).

[0049] Microorganisms producing the culture described above shall no berestricted and include Esherichia coli W3110 trp AE1 trp R tna A(pSC101-trp 115) (FERM P-17433; domestically deposited to the followinginstitute effective as of Jun. 28, 1999) (refer to Japanese PatentApplication Laid-Open No. 140891/1984; this bacterial strain wastransferred to National Institute of Bioscience and Human-TechnologyNational Institute of Advanced Industrial Science and Technology, 1-3,Higashi 1-chome, Tsukuba city, Ibaraki prefecture 305-8566, Japaneffective as of Feb. 7, 2001 to obtain a deposit number of FERM BP-7444)and in addition thereto, publicly known recombinant microorganism usedfor a producing L-tryptophan described above.

[0050] A method for producing a recombinant organism having the activitydescribed above (incorporating a gene coding a tryptophan synthaseoriginating in microorganisms into a suitable vector and then subjectingthe respective hosts to transformation) is known to a person having anaverage skill in the art, and capable of being given as patentliteratures which can be referred to are, for example, Japanese PatentApplication Laid-Open No. 255082/1990, Japanese Patent ApplicationLaid-Open No. 265892/1989 and Japanese Patent Application Laid-Open No.211319/1994. Accordingly, a microorganism which can producecorresponding L-7-substituted tryptophan from 7-substituted indole ofFormula (II) described above and L-serine shall not be restricted tomicroorganisms in which a gene coding a tryptophan-synthesizing enzymeused for a specific genus and recombination has specific origin.However, capable of being given as a suitable organism is Esherichiacoli W3110 trp AE1 trp R tna A (pSC101-trp 115) (FERM BP-7444). Thismicroorganism is lacking in a tryptophanase activity, and a plasmidcontaining a tryptophanase operon can very stably be present in a hostfungus and therefore is easily handled. The present invention shall beexplained below by citing the above FERM BP-7444 strain (hereinafterreferred to as an AGX-1757 strain) for the purpose of simplifyexplanation. In such biochemical conversion step, a R_(1a) group in7-substituted indole of the Formula (II) represents preferably theformula —(CH₂)_(p)—R_(b)′ considering chemical treatment thereafter.

[0051] A cultured substance is prepared by culturing the AGX-1757 strainon a culture medium and a culture condition which are usually used forculturing E. coli. 7-Substituted indole of Formula (II) and L-serine maybe contained in advance in a culture medium, and in general, amicroorganism is preferably added to a cultured substance up to a logphase or a steady phase and then incubated. The culture described abovecan be carried out on a culture medium containing a carbon source, anitrogen source, inorganic salts and the like which are conventionallyknown and usually used. Used as the carbon source are, for example,glucose, glycerol, fructose, sucrose and blackstrap molasses, and usedas the nitrogen source are, for example, ammonia, ammonium sulfate,ammonium chloride and ammonium nitrate. They each are used alone or in amixture. Further, used as the inorganic salts are, for example,potassium monohydrogenphosphate, potassium dihydrogenphosphate andmagnesium sulfate. In addition thereto, capable of being added to theculture medium are nutrients including peptone, meat extract, enzymeextract, corn steep liquor, cazaminoic acid and various vitamins such asbiotin and thiamin.

[0052] Culturing can be carried out usually at a culture temperature of20 to 50° C. under an aerobic condition of aerobic stirring and shaking.The pH in the middle of culturing can be set to the vicinity of 5 to 10,preferably 7 to 8, and the pH during culturing can be controlled byadding an acid or an alkali.

[0053] In general, 7-substituted indole of Formula (II) and L-serine arepreferably added to a culture at the same time, but it shall not berestricted thereto. When a pH of a culture liquid exceeds 8.0 or afterit is controlled to such level, the raw materials to be reacted arepreferably added. Incubation after adding them can be carried out at thesame temperature as the culture temperature described above whileaerobically stirring. An addition proportion of 7-substituted indole ofFormula (II) to L-serine is 1:5 to 2:1, preferably 1:1.1 in terms of amole ratio. In this case, a concentration of 7-substituted indole addedshall not be restricted as long as it does not exert an adverse effecton the intended conversion reaction, and it is preferably 5 to 50 mM.

[0054] In another method, mycelium are taken from the culture describedabove, and these mycelium themselves or a crude enzyme preparationprepared from the mycelium are sealed in a system by making use of amembrane to react 7-substituted indole with L-serine described above.

[0055] L-7-substituted tryptophan produced by the conversion reactiondescribed above can be obtained in the form of a pure product by aconventionally known separation-purifying method of tryptophan.

[0056] The foregoing process for producing L-7-substituted tryptophannot only constitutes one embodiment of the present invention but also isone essential step in producing the compound of Formula (I).

[0057] The compound of Formula (I) is produced via a chemical syntheticconversion reaction in which a reaction method itself is publicly knownusing the L-7-substituted tryptophan produced above as a startingmaterial. The typical conversion reaction is shown in the followingreaction scheme.

[0058] A reaction for reducing a compound (1) to a compound (2) iscarried out in a solvent using a reducing agent such as diborane,lithium aluminum hydride and the alkoxy complexes, or sodium borohydrideto which transition metal salts, aluminum chloride, boron trifluoride,phosphorus oxychloride, iodine or carboxylic acid (for example, aceticacid and trifuoroacetic acid) is added. The present reducing reaction iscarried out in a solvent including ethers such as diethyl ether,tetrahydrofuran, dimethoxyethane, dioxane and diglyme, toluene,chloroform and methylene chloride, and the solvent is suitably selecteddepending on the kind of the reducing agent used. The reactiontemperature is varied depending on the kind of the reducing agent, andit is usually about 0° C. to about 160° C., preferably about 10° C. toabout 80° C.

[0059] A protective group for an amino group of the compound (2)includes ethoxycarbonyl, t-butoxycarbonyl, acetyl, benzoyl,trifluoroacetyl, benzyloxycarbonyl and phthaloyl. The reaction can becarried out according to a conventional method, and it is carried out byreacting with a reagent of the intended protective group in a suitablesolvent. The solvent is selected depending on the kind of the protectivegroup, and the reaction temperature is usually about −20° C. to about200° C., preferably about 0° C. to about 150° C.

[0060] A hydroxyl group of the compound (3) is halogenated by directhalogenation using a halogenating agent such as thionyl chloride andthionyl bromide or direct halogenation in the presence oftriphenylphosphine. Also, the hydroxyl group of the compound (3) is onceturned into sulfonyloxy to form a compound (3′), and it is furtherreacted with alkali halide, whereby it can be halogenated as well. Thesereactions are carried out usually in suitable solvents, and the solventsused have to be suitably selected depending on the kind of the reactionreagents. They include, for example, aromatic hydrocarbons such asbenzene, toluene and xylene, ethers such as diethyl ether,tetrahydrofuran and dioxane, halogenated hydrocarbons such as methylenechloride and chloroform, ketones such as acetone and methyl ethylketone, ethyl acetate, acetonitrile, dimethylforamide anddimethylsulfoxide. These solvents each are used alone or in a mixture oftwo or more kinds thereof.

[0061] The present reaction is carried out, if necessary, in thepresence of a base, and specific examples of the base include alkalihydroxides such as sodium hydroxide and potassium hydroxide, alkalicarbonates such as sodium carbonate and potassium carbonate, alkalibicarbonates such as sodium bicarbonate and potassium bicarbonate andorganic bases such as triethylamine, tributylamine,diisopropylethylamine and N-methylmorpholine. The reaction temperatureis usually about −20° C. to about 200° C., preferably about 0° C. toabout 150° C.

[0062] A reducing reaction of a compound (5) in which halogen issubstituted with hydrogen from a compound (4) is carried out by reactingthem in a suitable solvent in the presence of a palladium on carboncatalyst and hydrogen or a hydrogen-donating substance such as ammoniumformate and cyclohexene. Used as the solvent are, for example, alcoholssuch as ethanol and methanol, water, acetic acid, tetrahydrofuran,dioxane, acetone, ethyl acetate, acetonitrile and dimethylforamide. Thereaction temperature is usually about 0° C. to about 150° C., and thereaction is carried out at an atmospheric pressure or under appliedpressure.

[0063] A deprotecting reaction of the compound (5) is carried out byhydrolysis, hydrogenolysis or reaction with hydrazine (the protectivegroup is phthaloyl). Deprotecting by hydrolysis can be carried out by aconventional method, and it is carried out, for example, by bringing theprotective group into contact with water in a suitable solvent on anacid or basic condition. Used as the solvent are, for example, alcoholssuch as ethanol and methanol, water, tetrahydrofuran, dioxane, acetone,acetonitrile, dimethylforamide or a mixed solvent thereof. Specificexamples of the acid include mineral acids such as hydrochloric acid,hydrobromic acid, hydroiodic acid and sulfuric acid and organic acidssuch as formic acid, acetic acid, trifluoroacetic acid,p-toluenesulfonic acid and methanesulfonic acid. Specific examples ofthe base include alkali hydroxides such as sodium hydroxide andpotassium hydroxide and alkali carbonates such as sodium carbonate andpotassium carbonate. The reaction temperature is usually about 0° C. toabout 150° C.

[0064] Deprotecting by hydrogenolysis can be carried out by aconventional method, and to be specific, it is carried out by the methoddescribed above. Elimination of a phthaloyl group is carried out usuallyby reacting with hydrazine hydrate in a solvent including alcohols suchas ethanol and methanol. The reaction temperature is usually about 50°C. to about 100° C.

[0065] An exchange reaction of R₁ is carried out by reaction of a7-hydroxyindole derivative [obtained by catalytically reducing R_(1a)which is a benzyl group allowed to be substituted(—(CH₂)_(m)—CHR_(a)′R_(aa)′ (wherein m=0, R_(a)′=a hydrogen atom,R_(aa)′=a phenyl group allowed to be substituted) or a benzhydryl groupallowed to be substituted (—(CH₂)_(m)—CHR_(a)′R_(aa)′ (wherein m=0,R_(a)′=R_(aa)′=a phenyl group allowed to be substituted)] with acorresponding alkyl derivative. This reaction is carried out in asolvent or in the absence of a solvent. The solvent used has to besuitably selected depending on the kind of the raw material compounds,and it includes, for example, aromatic hydrocarbons such as benzene,toluene and xylene, ethers such as diethyl ether, tetrahydrofuran anddioxane, halogenated hydrocarbons such as methylene chloride andchloroform, alcohols such as ethanol and isopropanol, ketones such asacetone and methyl ethyl ketone, ethyl acetate, acetonitrile,dimethylforamide, dimethylsulfoxide and ethylene glycol. These solventseach are used alone or in a mixture of two or more kinds thereof. Thepresent reaction is carried out, if necessary, in the presence of abase, and specific examples of the base include alkali hydroxides suchas sodium hydroxide and potassium hydroxide, alkali carbonates such assodium carbonate and potassium carbonate, alkali bicarbonates such assodium bicarbonate and potassium bicarbonate and organic bases such astriethylamine, tributylamine, diisopropylethylamine andN-methylmorpholine. The reaction temperature is usually about −20° C. toabout 200° C., preferably about 20° C. to about 150° C.

[0066] The present invention shall further be explained with referenceto specific examples.

EXAMPLE 1 Production of L-7-benzyloxytryptophan

[0067]

[0068] (i) Culture of E. coli

[0069] An Esherichia coli AGX-1757 (FERM BP-7444) strain was cultured onthe following conditions using a jar fermentor having a volume of 3liter.

[0070] Conditions: Cazaminoic acid 2.5 g Ammonium sulfate 10 g Enzymeextract 1 g Monopotassium phosphate 2 g Magnesium sulfate 7 hydrate 1 gFerrous sulfate 7 hydrate 10 mg Defoaming agent Adekapulronic 75 μlDistilled water 900 ml 60% Glucose aqueous solution 100 ml pH 7.0

[0071] Culturing was carried out at a temperature of 37° C. Aerobicstirring was carried out during culturing so that a dissolved oxygenconcentration does not become rate-determining. A pH of the culturesolution was reduced until 20 hours since starting culture, andtherefore a sodium hydroxide solution was always added to maintain pH at7.0.

[0072] (ii) Conversion Reaction

[0073] When 20 hours passed since starting the culturing describedabove, glucose contained in the culture medium spent up, and a rise inthe pH and the dissolved oxygen concentration was observed. When the pHexceeded 8.0, 10 g (12.5% methanol solution) of 7-benzyloxyindole and5.2 g (25% aqueous solution) of L-serine were added to the culturesolution to carry out the conversion reaction while aerobically stirringat 37° C. After reaction for 4 hours, a sodium hydroxide aqueoussolution was added to control the pH to 11.3, and the mycelium wasseparated by centrifugal filtering. A sulfuric acid solution was addedto this filtrate to control the pH to 7, and the resulting precipitatewas filtered and dried under reduced pressure to obtain a crude crystalof L-7-benzyloxytryptophan. This crude crystal was suspended in water,and a sodium hydroxide aqueous solution was added thereto to dissolveit. Then, sulfuric acid was added similarly to precipitate it. Thisprecipitate was filtered and dried to obtain 9.7 g of the captionedcompound.

[0074] Melting point: 213 to 218° C. (decomposed)

[0075] Angle of rotation: +1.80 (c1.0, 0.05M NaOH)

[0076] FABMS (m/z): 311 [(M+H)⁺]

[0077] NMR(DMSO-d6)δ 2.89 (1H, dd, J=8.8, 15.0 Hz), 5.25 (2H, s), 6.73(1H, d, J=7.7 Hz), 6.88 (1H, dd, J=7.7, 7.7 Hz), 7.14 (1H, dd, J=7.7,7.7 Hz), 7.14 (1H, d, J=7.7 Hz), 7.34 (1H, d, J=7.7 Hz), 7.40 (2H, dd,J=7.0, 7.7 Hz), 7.55 (2H, d, J=7.0 Hz), 10.97 (1H, broad)

[0078] 2H is superposed on 3.3 ppm DHO.

[0079] In analysis using CHIRALPAK AD (mobile phase:n-hexane/ethanol/trifluoroacetic acid=90/10/0.2), only a peak of an Lisomer was detected, and a D isomer was not observed.

EXAMPLE 2 Production of L-7-diethylaminocarbonylmethyloxytryptophan

[0080]

[0081] The conversion reaction described in Example 1 was substantiallyrepeated to obtain a reaction liquid, except that 0.5 g (10% methanolsolution) of 7-diethylaminocarbonylmethyloxyindole and 5.0 g (25%aqueous solution) of L-serine were substituted for 7-benzyloxyindole andL-serine used in Example 1 and that used was a culture solution preparedby controlling a pH of one liter of a culture solution obtained afterculturing for 22 hours to 9.0 by adding aqueous ammonia. A supernatantobtained by centrifuging this solution was passed through a columnfilled with Diaion HP-20 (manufactured by Mitsubishi Chemical Co., Ltd.)and adsorbed thereon, and then it was eluted by 30% methanol to obtain afraction corresponding to the product, which was dried up. The driedmatter was dissolved in 50% acetonitrile, and then the solution wasfractioned through fractional HPLC [column Inertsil PREP-ODS (GASUKUROKOGYO); mobile phase 20% acetonitrile, isocratic eluted, detection:UV254 nm] to obtain a product-containing fraction, and it was dried upto obtain 0.5 g of the intended compound. The compound thus obtained wasidentified to be 7-diethylaminocarbonylmethyloxytryptophan by means of¹H-NMR and FAB mass spectrum.

EXAMPLE 3 Preparation of(S)-7-benzyloxy-3-(2-amino-3-hydroxypropyl)indole

[0082]

[0083] L-7-Benzyloxytryptophan 50 g was suspended in 400 ml oftetrahydrofuran (THF), and 15 g of sodium borohydride was added theretoat 0° C., followed by dropwise adding a solution of 40 g of iodine inTHF (100 ml) in 40 minutes. After refluxing for 2 hours by heating, thesolution was cooled to 0° C., and 25 ml of methanol and 500 ml ofhydrochloric acid (2 mol/l) were dropwise added thereto. Then, thesolvent was evaporated under reduced pressure until the solution amountbecame about a half. The solution was cooled to 0° C., and 600 ml of asodium hydroxide aqueous solution (2 mol/1) was dropwise added thereto.The solution was extracted with 1000 ml of ethyl acetate, and theorganic layer was washed with a saturated brine. The resulting extractwas dried over anhydrous sodium sulfate, and then the solvent wasevaporated under reduced pressure to give 45.2 g of the title compound.

[0084] FABMS(m/z): 297 [(M+1)]

[0085] NMR(CDCl3) δ: 2.70 (1H, dd, J=8.0, 14.3 Hz), 2.92 (1H, dd, J=4.7,14.3 Hz), 3.25 (1H, m), 3.42 (1H, dd, J=7.0, 10.6 Hz), 3.68 (1H, dd,J=2.3, 10.6 Hz), 5.20 (2H, s), 6.73 (1H, d, J=7.7 Hz), 6.9-7.1 (2H, m),7.22 (1H, d, J=7.7 Hz), 7.3-7.5 (5H, m), 8.29 (1H, broad)

EXAMPLE 4 Preparation of(S)-7-benzyloxy-3-(3-hydroxy-2-phthalimidepropyl)indole

[0086]

[0087] (S)-7-Benzyloxy-3-(2-amino-2-hydroxypropyl)indole 45.2 g wasdissolved in 150 ml of N-dimethylformamide (DMF), and 25 ml oftriethylamine and 29 g of phthalic anhydride were added thereto,followed by stirring the solution at 130° C. for 2.5 hours. The solutionwas cooled to 0° C. and then diluted with 300 ml of toluene, and it waswashed with 200 ml of hydrochloric acid (0.1 mol/l). The separatedaqueous layer was further extracted twice with 100 ml of toluene. Ethylacetate 200 ml was added to the combined toluene layer, and the organicsolution was washed successively with twice a 10% sodium thiosulfateaqueous solution, a sodium hydrogencarbonate aqueous solution, and asaturated brine. The extract thus obtained was dried over anhydroussodium sulfate, and then the solvent was evaporated under reducedpressure until the whole amount was reduced to 400 ml. This solution wasleft standing one night to collect by filtration the resulting crystal,and then it was washed with 300 ml of toluene and 300 ml of hexane anddried to give 35.5 g of the title compound.

[0088] FABMS(m/z): 426 [(M)⁺]

[0089] NMR(CDCl3)δ: 3.32 (1H, dd, J=7.3, 15.0 Hz), 3.39 (1H, dd, J=7.3,15.0 Hz), 3.94 (1H, dd, J=3.3, 12.1 Hz), 4.10 (1H, dd, J=6.7, 12.1 Hz),4.76 (1H, dddd, J=3.3, 6.7, 7.3, 7.3 Hz), 5.17 (2H, s), 6.70 (1H, d,J=8.1 Hz), 7.01 (1H, dd, J=8.1, 8.1 Hz), 7.04 (1H, d, J=2.6 Hz), 7.3-7.5(6H, m), 7.70 (2H, dd, J=2.9, 5.5 Hz), 7.80 (2H, dd, J=2.9, 5.5 Hz),8.24 (1H, broad)

EXAMPLE 5 Preparation of (S)-7-benzyloxy-3-(2-phthalimide-3-p-toluenesulfonyloxypropyl)indole

[0090]

[0091] (S)-7-Benzyloxy-3-(3-hydroxy-2-phthalimidepropyl)indole 3.8 g wasdissolved in 9.5 ml of pyridine, and 2.1 g of p-toluenesulfonyl chloridewas added thereto, followed by stirring the solution at room temperaturefor 2 hours. The solution was diluted with 50 ml of ethyl acetate, andit was washed successively with water, three times with hydrochloricacid (2 mol/l) and a saturated brine. The extract thus obtained wasdried on anhydrous sodium sulfate, and then the solvent was evaporatedunder reduced pressure to give 4.68 g of the title compound.

[0092] FABMS (m/z): 580 [(M)⁺]

[0093] NMR(CDCl3) δ: 2.32 (3H, s), 3.24 (1H, dd, J=8.1, 15.2 Hz), 3.30(1H, dd, J=8.1, 15.2 Hz), 4.32 (1H, dd, J=3.7, 10.3 Hz), 4.78 (1H, dd,J=10.3, 10.3 Hz), 4.8-4.9 (1H, m), 5.15 (2H, s), 6.68 (1H, d, J=7.7 Hz),6.92 (1H, d, J=2.5 Hz), 6.97 (1H, dd, J=7.7, 7.7 Hz), 7.10 (1H, d, J=7.7Hz), 7.10 (1H, d, J=8.0 Hz), 7.13 (1H, d, J=8.0 Hz), 7.3-7.5 (5H, m),7.61 (2H, d, J=8.4 Hz), 7.6-7.8 (4H, m), 8.19 (1H, broad)

EXAMPLE 6-1 Preparation of (S)-7-benzyloxy-3-(3-iodo-2-phthalimidepropyl)indole

[0094]

[0095] (S)-7-Benzyloxy-3-(3-hydroxy-2-phthalimidepropyl)indole 35.45 gwas dissolved in 177 ml of THF, and 14.3 g of imidazole, 26.4 g oftriphenylphosphine and 42.6 g of iodine were added thereto, followed bystirring the solution at 60° C. for 3 hours. The reaction solution wascooled down and then diluted with 1,060 ml of ethyl acetate, and it waswashed successively with a saturated sodium hydrogencarbonate aqueoussolution, twice a 10% sodium thiosulfate aqueous solution and asaturated brine. The extract thus obtained was dried over anhydroussodium sulfate and then concentrated under reduced pressure, and 350 mlof methanol was added to the resulting residue. The solution was heatedto reflux at 80° C., and a white crystal formed was collected byfiltration and washed with 106 ml of methanol, followed by drying toobtain 30.06 g of the title compound.

[0096] FABMS (m/z): 537 [(M+H)⁺]

[0097] NMR (CDCl3) δ: 3.44 (2H, d, J=8.4 Hz), 3.56 (1H, dd, J=4.8, 10.4Hz), 4.03 (1H, dd, J=10.4, 10.4 Hz), 4.84 (1H, ddt, J=4.8, 8.4, 10.4Hz), 5.16 (2H, s), 6.70 (1H, d, J=7.7 Hz), 6.99 (1H, d, J=2.6 Hz), 7.01(1H, dd, J=7.7, 7.7 Hz), 7.26 (1H, d, J=8.1 Hz), 7.3-7.5 (5H, m), 7.69(2H, dd, J=2.9, 5.5 Hz), 7.80 (2H, dd, J=2.9, 5.5 Hz), 8.23 (1H, broad)

EXAMPLE 6-2 Preparation of (S)-7-benzyloxy-3-(3-iodo-2-phthalimidepropyl)indole

[0098]

[0099](S)-7-Benzyloxy-3-(2-phthalimide-3-p-toluenesulfonyloxypropyl)indole4.68 g was dissolved in 10 ml of DMF, and 2.0 g of sodium iodide wasadded thereto, followed by stirring the solution at 120° C. for 2 hours.The reaction solution was cooled down and then diluted with 30 ml oftoluene, and it was washed successively with water and a saturatedbrine. The extract thus obtained was dried over anhydrous sodium sulfateand then concentrated under reduced pressure. The residue was purifiedby silica gel column chromatography to give 3.21 g of the titlecompound.

EXAMPLE 7 Preparation of (R)-7-benzyloxy-3-(2-phthalimidepropyl)indole

[0100]

[0101] A 10% palladium on carbon catalyst (content 50%) 4.00 g wassuspended in 200 ml of DMF, and 20.00 g of(S)-7-benzyloxy-3-(3-iodo-2-phthalimidepropyl)indole was added thereto.Added while stirring were 8.00 g of potassium carbonate and 6.00 g ofammonium formate, the suspension was heated in an oil bath of 85° C. forone hour. After cooling down, the catalyst was filtered off, and thefiltrate was diluted with ethyl acetate. Then, water was added thereto,and the organic layer was separated. The organic layer was washed inorder with a 10% sodium thiosulfate aqueous solution, a sodium hydroxideaqueous solution, hydrochloric acid and a saturated sodium chlorideaqueous solution, and then it was dried, filtered and concentrated underreduced pressure, whereby 12.16 g of the title compound was obtained inthe form of a yellow amorphous solid shaped substance.

[0102] FABMS (m/z): 410 [(M)⁺]

[0103] NMR (CDCl3) δ: 1.54 (3H, d, J=7.0 Hz), 3.23 (1H, dd, J=7.3, 14.7Hz), 3.46 (1H, dd, J=8.5, 14.7 Hz), 4.77 (1H, ddd, J=7.0, 7.3, 8.5 Hz),5.12 (2H, s), 6.68 (1H, d, J=7.7 Hz), 6.96 (1H, d, J=2.6 Hz), 7.00 (1H,dd, J=7.7, 8.1 Hz), 7.29 (1H, d, J=8.1 Hz), 7.30-7.45 (5H, m), 7.62-7.66(2H, m), 7.73-7.77 (2H, m), 8.18 (1H, broad)

EXAMPLE 8 Preparation of (R)-7-hydroxy-3-(2-phthalimidepropyl)indole

[0104]

[0105] (R)-7-Benzyloxy-3-(2-phthalimidepropyl)indole 12.16 g wasdissolved in 400 ml of ethanol, and a water 100 ml suspension of a 10%palladium on carbon catalyst (content 50%) 4.00 g prepared separatelywas added thereto. Ammonium formate 6.00 g was added thereto whilestirring, and the suspension was heated in an oil bath of 95° C. for onehour. After cooling down, the catalyst was filtered off, and thefiltrate was concentrated under reduced pressure. Acetone was added tothe residue, and a deposited salt was filtered off. The filtrate wasconcentrated under reduced pressure, whereby 10.27 g of a residuecontaining the title compound was obtained.

[0106] FABMS(m/z): 320 [(M)⁺]

[0107] NMR (CDCl3) δ: 1.55 (3H, d, J=7.0 Hz), 3.23 (1H, dd, J=7.0, 14.7Hz), 3.46 (1H, dd, J=8.8, 14.7 Hz), 4.77 (1H, ddd, J=7.0, 7.0, 8.8 Hz),5.71 (1H, broad), 6.54 (1H, d, J=7.7 Hz), 6.90 (1H, dd, J=7.7, 8.1 Hz),6.93 (1H, d, J=2.2 Hz), 7.23 (1H, d, J=8.1 Hz), 7.60-7.64 (2H, m),7.71-7.76 (2H, m), 8.20 (1H, broad)

EXAMPLE 9 Preparation of(R)-7-diethylaminocarbonylmethoxy-3-(2-phthalimidepropyl)indole

[0108]

[0109] A residue 10.27 g containing(R)-7-hydroxy-3-(2-phthalimidepropyl)indole was dissolved in 200 ml ofacetone, and 8.00 g of potassium carbonate was added thereto. Afteraddition of N,N-diethylchloroacetamide 15.5 ml, the solution was heatedto reflux in an oil bath of 80° C. for 3 hours. After cooling down 200ml of water was added, and a deposited crystal was collected byfiltration and washed with water, whereby 9.56 g of the title compoundwas obtained in the form of a pale yellowish white substance.

[0110] FABMS(m/z): 433 [(M)⁺]

[0111] NMR (CDCl3) δ: 1.14 (3H, d, J=7.1 Hz), 1.19 (3H, d, J=7.1 Hz),1.54 (3H, d, J=7.0 Hz), 3.24 (1H, dd, J=7.3, 14.7 Hz), 3.32 (2H, q,J=7.1 Hz), 3.41 (2H, q, J=7.1 Hz), 3.45 (1H, dd, J=8.1, 14.7 Hz), 4.75(2H, s), 4.77 (1H, ddd, J=7.0, 7.3, 8.1 Hz), 6.62 (1H, d, J=7.7 Hz),6.97 (1H, dd, J=7.7, 8.1 Hz), 7.00 (1H, d, J=2.6 Hz), 7.33 (1H, d, J=8.1Hz), 7.63-7.66 (2H, m), 7.73-7.76 (2H, m), 9.34 (1H, broad)

EXAMPLE 10 Preparation of(R)-7-diethylaminocarbonylmethyloxy-3-(2-aminopropyl)indole

[0112]

[0113] (R)-7-Diethylaminocarbonylmethyloxy-3-(2-phthalimidepropyl)indole10.27 g was suspended in 100 ml of ethanol, and 2.5 ml of hydrazinemonohydrate was added thereto. The suspension was heated to reflux in anoil bath of 85° C. for 3 hours. After cooling down, the resultingdeposited insoluble materials were filtered off, and the filtrate wasconcentrated under reduced pressure. Ethyl acetate and a sodiumhydroxide aqueous solution were added to the residue and distributed inliquid-liquid, and the organic layer was further washed with a sodiumhydroxide aqueous solution and dried. The solution was evaporated underreduced pressure. The resulting residue was slowly crystallized, anddried under reduced pressure, whereby 6.90 g of the title compound wasobtained in the form of a pale brownish white substance.

[0114] FABMS (m/z): 304 [(M+H)⁺]

[0115] NMR (CD30D) δ: 1.12 (3H, d, J=6.6 Hz), 1.17 (3H, t, J=7.1 Hz),1.24 (3H, t, J=7.1 Hz), 2.70 (1H, dd, J=7.8, 14.2 Hz), 2.81 (1H, dd,J=6.2, 14.2 Hz), 3.19 (1H, ddd, J=6.2, 6.6, 7.8 Hz), 3.43-3.49 (4H, m),4.89 (2H, s), 6.64 (1H, d, J=7.7 Hz), 6.91 (1H, t, J=7.7 Hz), 7.07 (1H,s), 7.19 (1H, d, J=8.1 Hz)

EXAMPLE 11 Preparation of(S)-7-benzyloxy-3-(2-t-butoxycarbonylamino-3-hydroxypropyl)indole

[0116]

[0117] (S)-7-Benzyloxy-3-(2-amino-3-hydroxypropyl)indole 2.53 g wasdissolved in 20 ml of 1,4-dioxane, and 20 ml of sodium hydroxide (1mol/l) was added thereto. After addition of di-tert-butylpyrocarboxylate 1.70 g, the mixture was stirred for 1.5 hour. Thesolution was diluted with 80 ml of ethyl acetate and then washed with 50ml of water. The separated aqueous layer was further extracted with 100ml of ethyl acetate, and the combined organic layer was washed with asaturated brine. The extract was dried over anhydrous sodium sulfate,and then the solvent was distilled off. The resulting residue waschromatographed on silica gel (hexane/ethyl acetate 1/1) to give 3.21 gof the title product.

[0118] FABMS (m/z): 396 [(M)⁺]

EXAMPLE 12 Preparation of(S)-7-benzyloxy-3-(2-t-butoxycarbonylamino-3-iodo)indole

[0119]

[0120] (S)-7-Benzyloxy-3-(2-t-butoxycarbonylamino-3-hydroxypropyl)indole3.21 g was dissolved in 50 ml of THF, and 1.10 g of imidazole, 4.23 g oftriphenylphosphine and 3.28 g of iodine were added thereto, followed bystirring the solution at room temperature for 25 minutes. A 10% sodiumthiosulfate aqueous solution 50 ml was added thereto. The solution wasstirred and extracted with 300 ml of ethyl acetate. The extract waswashed successively with 100 ml of a 10% sodium thiosulfate aqueoussolution was further added and distributed in liquid-liquid. The organiclayer was washed with 100 ml of a 10% sodium thiosulfate aqueoussolution and a saturated brine. The extract was dried over anhydroussodium sulfate and concentrated to dryness under reduced pressure, togive 6.13 g of a residue containing the title compound was obtained.

[0121] FABMS (m/z): 506 [(M)⁺]

COMPARATIVE EXAMPLE

[0122] In this experiment, investigated was conversion from7-benzyloxyindole to a corresponding tryptophan derivative usingcultured substances of E. coli K-12 (ATCCC 14948) and AGX-1757 (FERMP-17433) as a tryptophan-synthesizing enzyme-producing microorganism.

[0123] The experimental conditions and the results are shown belowrespectively. Culture medium: K-12 strain brain heart infusion culturemedium (Difco) AGX-1757 brain heart infusion culture medium containingoxytetracycline monohydrochloride (Wako Pure Chemical) of 10 mg/l

[0124] Culturing method:

[0125] Used was a 500 ml Erlenmeyer flask containing 50 ml of theculture medium having the composition described above.

[0126] A K12 strain grown on a slant was inoculated.

[0127] An AGX-1757 strain 200 μl of a froze culture stock wasinoculated.

[0128] Cultured at 32° C. in a rotary shaker of 240 rpm. Conversionsubstrate: 7-benzyloxyindole 125 g/l methanol solution L-serine 250 g/laqueous solution Cultured broth:

[0129] Cultured for 22 hours. OD 660 nm values were 9.82 in the case ofthe K-12 strain and 7.86 in the case of the AGX-1757 strain. The culturebroth had a pH of 8.54 in the case of the K-12 strain and 8.36 in thecase of the AGX-1757 strain.

[0130] Conversion Reaction:

[0131] Added to these flasks were 4 ml of a 7-benzyloxyindole solution,10 g/l and an L-serine solution, 10 g/l to carry out conversion reactionat 32° C. and 240 rpm.

[0132] Results:

[0133] A concentration of 7-benzyloxytryptophan contained in the culturesolution was analyzed every 2 hours up to 6 hours by means of HPLC.

[0134] Formation of 7-benzyloxytryptophan was not observed in the K-12strain. (Analysis sensitivity: 0.1 g/l or more)

[0135] In the AGX-1757 strain, formed were 2.33 g/l of7-benzyloxytryptophan in 2 hours, 4.45 g/l in 4 hours and 6.93 g/l in 6hours.

[0136] The results thereof are summarized in Table 2.

[0137] HPLC Conditions:

[0138] Column: Super-ODS (TOSO)

[0139] Elution: acetonitrile/water gradient elution

[0140] Temperature: 40° C.

[0141] Flow velocity: 2 ml/minute

[0142] Detection: UV 254 nm TABLE 2 Fungus strain Time (hour) 7BOT 7BOIK-12 2 0 10.2 4 0 9.86 6 0 10.1 AGX-1757 2 2.33 7.05 4 4.45 5.81 6 6.935.86

INDUSRIAL APPLICABILITY

[0143] According to the present invention, provided is an efficientprocess for producing a specific optically active7-substituted-3-(2-aminopropyl)indole compound. The above compound canbe used as a synthetic intermediate for some fixed medicines.Accordingly, the present invention can be utilized, for example, in themedicine production industry.

1. A process for preparing an optically active indole derivative of theFormula (I):

[wherein R₁ is a hydrogen atom, a lower alkyl group or a group selectedfrom the following (a) and (b): (a) a group of the formula—(CH₂)_(m)—CHR_(a)R_(aa) (wherein R_(a) is a hydrogen atom, a loweralkyl group, a lower alkoxycarbonyl group, a carboxyl group or a phenylgroup which may be substituted (a substituent thereof is a lower alkylgroup, a lower alkoxy group, a hydroxy group or a halogen atom); R_(aa)is a lower alkoxycarbonyl group, a carboxyl group or a phenyl groupwhich may be substituted (a substituent thereof is a lower alkyl group,a lower alkoxy group, a hydroxy group or a halogen atom); and mrepresents an integer of 0 to 3) and (b) a group of the formula—(CH₂)_(p)—R_(b) (wherein R_(b) is a lower alkanoyl group, a hydroxygroup, a cyano group or a mono- or di(lower alkyl)aminocarbonyl group,and p is an integer of 1 to 4); R₂ and R₃ are each a hydrogen atom, oreither of them is a hydrogen atom and the other is a lower alkanoylgroup, a lower alkoxycarbonyl group or an aralkyloxycarbonyl group, ortogether form a phthaloyl group; and R₄ is a hydrogen atom, a halogenatom, a hydroxy group, a lower alkylsulfonyloxy group or aphenylsulfonyloxy group which may be substituted (a substituent thereofis a lower alkyl group, a lower alkoxy group or a halogen atom)],comprising the steps of: a) reacting L- or DL-serine with 7-substitutedindole of the Formula (II):

[wherein R_(1a) is a group (c) or (d) shown below: (c) a group of theformula —(CH₂)_(m)—CHR_(a)′R_(aa)′ (wherein R_(a)′ is a hydrogen atom, alower alkyl group or a phenyl group which may be substituted (asubstituent thereof is a lower alkyl group, a lower alkoxy group, ahydroxy group or a halogen atom); R_(aa)′ is a phenyl group which may besubstituted (a substituent thereof is a lower alkyl group, a loweralkoxy group, a hydroxy group or a halogen atom); and m is an integer of0 to 3) and (d) a group of the formula —(CH₂)_(p)—R_(b)′ (wherein R_(b)′is a mono- or di(lower alkyl)aminocarbonyl group, and p is an integer of1 to 4)] in the presence of a tryptophan-synthesizing enzyme originatingin microorganisms to form 7-substituted-L-tryptophan of the Formula(III):

 (wherein R_(1a) is the same as described above), b) chemically reducinga carboxyl group of 7-substituted-L-tryptophan of Formula (III) thusformed to a methylol group, c) protecting and modifying, if necessary,an amino group, d) turning, if necessary, a hydroxy group of methyloldescribed above into sulfonyl to form a corresponding loweralkylsulfonyloxy compound or a phenylsulfonyloxy compound which may besubstituted (a substituent thereof is a lower alkyl group, a loweralkoxy group or a halogen atom), e) halogenating it, if necessary, withalkali halide or halogenating a hydroxy group of methylol describedabove directly or in the presence of triphenylphosphine to form acorresponding halide, f) converting, if necessary, the sulfonyloxycompound or the halide obtained in d) or e) to methyl by hydrogenatingin the presence of a palladium catalyst or using a metal hydride salt,g) eliminating, if necessary, the protective group of the amino group,and h) subjecting, if necessary, the substituent of R₁ to exchangereaction.
 2. The process as described in claim 1, wherein R_(1a) in7-substituted indole of Formula (II) is a group of the formula (c′)—(CH₂)_(m)—CHR_(a)″R_(aa)″ [wherein R_(a)″ is a hydrogen atom or aphenyl group which may be substituted (a substituent thereof is a loweralkyl group, a lower alkoxy group, a hydroxy group or a halogen atom);R_(aa)′ is a phenyl group which may be substituted (a substituentthereof is a lower alkyl group, a lower alkoxy group or a halogen atom);and m is an integer of 0 to 3].
 3. A process for preparing 7-substitutedL-tryptophan of the Formula (III):

(wherein R_(1a) is the same as shown below), characterized by reactingL- or DL-serine with 7-substituted indole of the Formula (II):

[wherein R_(1a) is a group (c) or (d) shown below: (c) a group of theformula —(CH₂)_(m)—CHR_(a)′R_(aa)′ (wherein R_(a)′ is a hydrogen atom, alower alkyl group or a phenyl group which may be substituted (asubstituent thereof is a lower alkyl group, a lower alkoxy group, ahydroxy group or a halogen atom); R_(aa)′ is a phenyl group which may besubstituted (a substituent thereof is a lower alkyl group, a loweralkoxy group, a hydroxy group or a halogen atom); and m is an integer of0 to 3) and (d) a group of the formula —(CH₂)_(p)—R_(b)′ (wherein R_(b)′is a mono- or di(lower alkyl)aminocarbonyl group, and p is an integer of1 to 4)] in the presence of a tryptophan-synthesizing enzyme originatingin microorganisms.
 4. An optically active indole derivative of theFormula (IV):

[wherein R₁ is a hydrogen atom, a lower alkyl group or a group selectedfrom the following (a) and (b): (a) a group of the formula—(CH₂)_(m)—CHR_(a)R_(aa) (wherein R_(a) is a hydrogen atom, a loweralkyl group, a lower alkoxycarbonyl group, a carboxyl group or a phenylgroup which may be substituted (a substituent thereof is a lower alkylgroup, a lower alkoxy group, a hydroxy group or a halogen atom); R_(aa)is a lower alkoxycarbonyl group, a carboxyl group or a phenyl groupwhich may be substituted (a substituent thereof is a lower alkyl group,a lower alkoxy group, a hydroxy group or a halogen atom); and m is aninteger of 0 to 3) and (b) a group of the formula —(CH₂)_(p)—R_(b)(wherein R_(b) is a lower alkanoyl group, a hydroxy group, a cyano groupor a mono- or di(lower alkyl)aminocarbonyl group, and p is an integer of1 to 4); R₂ and R₃ are each a hydrogen atom, or either of them is ahydrogen atom and the other is a lower alkanoyl group, a loweralkoxycarbonyl group or an aralkyloxycarbonyl group, or together form aphthaloyl group; and R₄′ is a halogen atom, a hydroxy group, a loweralkylsulfonyloxy group or a phenylsulfonyloxy group which may besubstituted (a substituent thereof is a lower alkyl group, a loweralkoxy group or a halogen atom)].
 5. A 7-substituted L-tryptophan of theFormula (III):

[wherein R_(1a) is a group (c) or (d) shown below: (c) a group of theformula —(CH₂)_(m)—CHR_(a)′R_(aa)′ (wherein R_(a)′ is a hydrogen atom, alower alkyl group or a phenyl group which may be substituted (asubstituent thereof is a lower alkyl group, a lower alkoxy group, ahydroxy group or a halogen atom); R_(aa)′ is a phenyl group which may besubstituted (a substituent thereof is a lower alkyl group, a loweralkoxy group, a hydroxy group or a halogen atom); and m is an integer of0 to 3) and (d) a group of the formula —(CH₂)_(p)—R_(b)′ (wherein R_(b)′is a mono- or di(lower alkyl)aminocarbonyl group, and p is an integer of1 to 4)].